Human carcinomas arise from normal epithelial cells through progressive relaxation of tumor suppressor checkpoints. HPV E6 oncogene can dominantly immortalize mammary epithelial cells, which otherwise exhibit a finite life span, providing a practical approach to identify tumor suppressor pathways inactivated during transformation of epithelial cells. This model revealed a crucial role for inactivation of p53 functions; however, selective elimination of p53 function did not lead to efficient immortalization, indicating the role of additional pathways. We have identified the human homologue of yeast transcriptional coactivator yADA3, as a novel E6 target. yADA3 is a critical component of coactivator complexes that link transcriptional activators to histone acetylation and basal transcriptional machinery. Reconstitution of mammalian nuclear receptor-mediated transcription in yeast revealed a requirement for yADA3, and associated proteins, yADA2 and yGCN5. Based on these studies, and selective binding of hADA3 to high-risk HPV E6 proteins and immortalizing E6 mutants, we hypothesize that hADA3 participates in transcriptional regulation of genes that maintain the normal state of epithelial cells. Loss or deregulation of hADA3 function, due to its interaction with HPV E6 or by other means, is likely to represent an important oncogenic mechanism in HPV-associated and other cancers. To address our hypotheses, we will generate antibodies to human ADA3 and ADA2, and examine their expression, and establish the presence of hADA3-containing histone acetylase complexes and their involvement in retinoid receptor-mediated gene transcription in epithelial cells. We will then define the domains of ADA3 that mediate its physical and functional interactions with activated retinoid receptors and histone acetylase complexes. We will examine whether E6 inactivates ADA3 function by displacement of binding partners or ADA3 degradation. Finally, we will determine the potential role of ADA3 in mediating cell differentiation and growth inhibitory functions, and assess the potential of dominant-negative ADA3 mutants to induce immortalization. Successful outcome of these studies should identify a novel tumor suppressor pathway whose components may provide new markers for early diagnosis of carcinomas, and targets for development of rational therapeutics.